Abstract

Specimens of bone marrow trephine biopsy (BMT) are transported and fixed in acetic acid–zinc–formalin fixative, decalcified
in 10% formic acid–5% formaldehyde and processed with other specimens to paraffin-wax embedding. Sections, 1-μm-thick, are
cut by experienced histotechnologists and used for haematoxylin and eosin, Giemsa, reticulin silver and other histological
stains. Further, all immunohistochemical procedures used in the laboratory, including double immunostaining, can be used on
these sections with no or minimal modifications. About 10 000 BMT specimens have been analysed using this procedure since
1997 and diseases involving the bone marrow have been classified successfully. More recently, standardised polymerase chain
reaction-based analysis and mRNA in situ hybridisation studies have been conducted. Excellent morphology with good antigen,
DNA and RNA preservation is offered by the Hammersmith Protocol.

A bone marrow trephine biopsy (BMT) is carried out to assess various haematological problems, particularly in those cases
where assessment of marrow cellularity, cell distribution and relationship between different cell types is crucial. It is
also important in disease processes that are focal in nature and in those that are associated with changes in bone, blood
vessels and marrow stroma. In some disorders, the pattern of infiltration provides additional prognostic information. Furthermore,
in cases where immunohistochemistry is required and where antigen expression has to be evaluated in spatial contexts, BMT
specimens have a major role. Their role is critical in cases of “dry tap”, where examination of an aspirate has been unsuccessful
owing to a fibrotic or infiltrative process.

In many laboratories standard tissue fixation and sectioning with routine decalcification results in BMT specimens which are
difficult to interpret and where immunohistochemical staining can be compromised. Tissue morphology is influenced by the quality
of the sample taken (haemorrhage and crush artefact can destroy even the most exquisitely processed sample), the length and
type of fixation, the tissue processing programme, the tissue sectioning and the quality of the staining. The presence of
bone in BMT specimens requires decalcification or the use of special blades that can cut un-decalcified bony tissues. Decalcification
represents an additional variable influencing the staining pattern, with deterioration in the preservation of cytoplasmic
granules and enzyme activity. More importantly, antigenicity in immunohistochemistry may be adversely affected, although the
adverse effects can be mitigated by the use of antigen-retrieval techniques.1 Resin embedding and cutting with tungsten carbide knives can result in improved cytomorphology. This, however, hinders immunohistochemical
procedures for several antigens and is an additional investment of time, expertise and effort for histopathology or haematopathology
laboratories.2

Routine formalin fixation followed by ethylene diamine tetra acetate (EDTA) or nitric acid decalcification are in common use,
but improved methods of fixation and decalcification have also been used to obtain improved tissue morphology in the paraffin-wax-embedded
BMT specimens as an alternate to resin embedding. Compared with formalin fixation, mercurial fixatives such as B5 and Zenker’s
fixative result in improved morphology.3 The use of these fixatives is, however, limited by the procedures associated with, and the expense of disposing of, the mercury-containing
solutions. In the UK, the Control of Substances Hazardous to Health stipulates that, whenever possible, processes using less
harmful substances should substitute for processes using toxic substances such as mercury. A more recent study compared B5
fixation with five other fixatives and showed that acetic acid–zinc–formalin (AZF) provided overall staining and morphological
detail comparable to B5 staining. The AZF fixative resulted in superior antigen preservation, and the RNA and DNA were also
found to be well preserved for in situ hybridisation (ISH) and molecular analysis.4

At the Hammersmith Hospital, London, UK, we have used AZF fixative for more than 10 000 BMT specimens since 1997. We standardised
a protocol for handling BMT specimens, where, after the initial fixation and decalcification, BMT specimens can be treated
similar to other biopsy specimens. We believe that this has been highly successful in terms of providing excellent cytomorphology
with haematoxylin and eosin (H&E) and Giemsa stains. Further, the results of other histochemical stains and immunohistochemistry
are comparable to those of other biopsy specimens.

BONE MARROW PROCESSING AND STAINING

Fixative

The AZF (zinc chloride, 12.5 g; concentrated formaldehyde, 150 ml; glacial acetic acid, 7.5 ml; and distilled water, to 1000
ml) is prepared in bulk and aliquotted into 20-ml universal containers with corrosive hazard labels. These aliquots are sent
to the haematology departments and other inpatient and clinic locations where BMTs are carried out. The haematologist is instructed
to place the freshly obtained BMT specimens directly into AZF fixative and transport it immediately to the histopathology
department, on the same day as the procedure.

Fixation

Once received in the laboratory, the BMT specimens are left to fix in AZF overnight. The next morning (after 20–24 h), the
solution is decanted (with a strainer) and the biopsy specimen is washed in distilled water for 30 min. Specimens from the
previous day, received in the histopathology department, having already been fixed in AZF overnight, are similarly treated.

Decalcification

The water is replaced by Gooding and Stewart’s decalcification fluid (10% formic acid and 5% formaldehyde). The biopsy specimens
are left to decalcify for about 6 h before being processed and embedded in paraffin wax, with procedures similar as for other
specimens.

Tissue sectioning and staining

Sections, 1-μm thick (microtome set for 1 μm sections), are cut from the paraffin-wax blocks with the routine rotary microtomes
in the laboratory. The sections are stained with H&E, Giemsa, Perl’s (iron) and reticulin (silver) stains. Additional unstained
sections are placed on poly-l-lysine-coated slides for immunostaining as necessary.

Immunohistochemistry

Immunohistochemistry panels are used based on the clinical diagnosis, and the required number of sections for each panel (including
those required for controls) is cut at the time of initial sectioning. Sections, 1-μm thick, are placed on poly-l-lysine-coated glass slides. The dewaxed sections are subjected to antigen-retrieval procedures as per the requirements for
different antigens. Table 1 gives the details of the antigen-retrieval procedures and other details of the individual protocols.
The antigen localisation is carried out using Super Sensitive TM Immunohistochemistry Detection System (Biogenex, California,
USA), with diaminobenzidine as the final substrate. The automated system developed by Biogenex has been used in most cases.
Immunostaining is carried out on the automated system on most occasions and manually on some occasions. We also carry out
sequential double immunostaining using previously described methods.5 For the second procedure, we use vector blue, amino ethylcarbazole or diaminobenzidine as chromogens.

Antibodies used on bone marrow trephine biopsy specimens at the Hammersmith Hospital

In situ hybridisation

ISH of mRNA is carried out using κ and λ Probe ISH Kit and Epstein–Barr Virus (EBV) Probe ISH Kit according to the manufacturer’s
instructions (Vision Biosystems Newcastle upon Tyne, UK; Novocastra, Newcastle upon Tyne, UK). For the purposes of probe penetration,
proteinase-K digestion is used before hybridisation. The procedure includes hybridisation for 4 h at 37°C with fluorescein-conjugated
oligonucleotide probes. After hybridisation and post-hybridisation washes, probe localisation is carried out using rabbit
F(ab′) anti-fluorescein isothiocyanate or alkaline phosphatase. Nitro blue tetrazolium or 5-bromo-4-chloro-3-indolyl phosphate
is used as substrate to produce a blue–black signal. To ensure optimal mRNA preservation in a given case, a poly-dT probe
(instead of the light chain or the EBV probe) on a separate section of the paraffin-wax block is used as a control.

DNA extraction and PCR

The protocol followed for the BMT specimens is the same as that previously used for paraffin-wax sections of lymphoreticular
tissues. Two 15-μm-thick paraffin-wax sections are cut from the paraffin-wax block on a rotary microtome into a 2 ml Eppendorf
tube. Precautions are taken to avoid tissue contamination from other cases. DNA is extracted using the Qiagen DNA Tissue Kit
(Gmbh, Frankfurt, Germany). The procedure includes dewaxing, followed by overnight proteinase-K digestion at 55°C in a water
bath. After addition of absolute ethyl alcohol, the digests are passed through a DNeasy Mini spin column. The DNA is then
eluted from the column with 100 μl of TRIS–EDTA buffer (pH 8.0). The quality of amplifiable DNA is assessed using primers
designed to amplify a 200 bp fragment of the β-actin gene (forward primer 5′CAACTACTCGGGAGGCTGAG3′ and reverse primer 5′GCCCCTGGTAGTTTTTCACA3′).
Samples are then subjected to other PCR-based molecular diagnostic assays. For the purpose of this paper, BMT specimens were
tested for immunoglobulin heavy chain rearrangement with primers directed at the variable heavy (FR3) region (5′ACACGGCGTGTGTGATATTACTGT3′)
and the JH regions (5′TGAGGAGACGGTGACCAGGATCCCTTGGCCCCAG3′). The samples were subjected to 40 cycles of amplification on a
PCR protocol of 30 s at 94°C, 30 s at 52°C and 30 s at 72°C, after an initial denaturation for 5 min at 94°C. The analysis
ended with a final extension at 72°C for 10 min. The PCR products were run on a 3% agarose gel, stained with ethidium bromide
and photographed under UV illumination. Cases with clonal rearrangements resulted in an amplicon of around 100 bp.

RESULTS AND COMMENTS

For optimal interpretation of BMT specimens, a combination of excellent cytomorphology, provision to use the full range of
immunohistochemical stains, preservation of mRNA to carry out ISH analyses and the ability to extract DNA of a quality sufficient
to carry out PCR-based analyses are all crucial. Although it is generally accepted that resin embedding and semithin sections
provide the best morphology, there have been questions on preservation of antigens and nucleic acids.2,6 Several papers deal with these issues and confirm adequate antigen and DNA preservation in resin-embedded BMT specimens.7,8,9,10 However, RNA preservation and suitability for ISH analyses of mRNA is not clear. The main criticism of this procedure is
the requirement of specialised technology and skill and the additional costs associated with it.

The principles of our approach are as follows:

Excellent morphology without compromising preservation of antigens and nucleic acids

Preservation of antigen and nucleic acid

Simple and integrated, with standard processing schedules

Inexpensive and non-toxic.

Optimal morphology without compromising preservation of antigens and nucleic acids

Our primary intention was to standardise a protocol for processing BMT specimens, which would result in cytomorphology of
a quality comparable to that of resin-embedded semithin sections and be suitable for a wide range of immunohistochemical staining.
With this purpose, we embarked on AZF fixative, which is formalin based and contains the nuclear mordant zinc. We are able
to cut 1-μm-thick sections providing excellent morphology (fig 1). To hasten the decalcification process, we use a short period
of formic acid decalcification.

EDTA, which is widely used for processing of BMT specimens, is slow and takes a minimum of 24 h (although application of an
ultrasonic bath can help hasten this process).11 Although some laboratories report better success at DNA amplification on samples decalcified with EDTA, this is variable.12 On the other hand, acid decalcification with hydrochloric acid, nitric acid or formic acid is faster, but is believed to
result in some destruction of antigens and tissue architecture. Short periods of acid decalcification, however, do not seem
to have a deleterious effect on molecular diagnostics, including fluorescence in situ hybridisation and comparative genomic
hybridisation studies.13,14

To obtain better morphology, fixatives like Bouin’s, mercury-containing solutions such as Zenker’s fixative and B5 have been
used. Some of these are, however, said to have a deleterious effect on preservation of antigens and nucleic acids, and mercurial
fixatives pose problems related to disposal.15,16 Although the morphology is not comparable to that of semithin sections, formalin fixation provides superior antigen and DNA
preservation as compared with picric acid or mercury-based fixative.16–18 Furthermore, formalin-fixed trephine sections provide adequate RNA preservation as seen in the few published studies on mRNA
ISH.19,20 Thus, formalin fixation seems to be a good “compromise” fixative for processing of BMT specimens. The main criticism has
been the suboptimal morphology as compared with resin-embedded sections.

Zinc-containing fixatives have been reported to be superior to formalin in terms of antigen preservation for immunohistochemistry
and the recent study by Bonds et al4 clearly shows the superiority of AZF over other fixatives for preservation of morphology, antigens, RNA and DNA.4,21

The mechanism by which zinc interacts with proteins and nucleic acids is unclear and is currently part of our research programme
in Imperial College. Formalin seems to be necessary for optimal morphology as our modified zinc-based fixative (Z7) still
results in some tissue shrinkage, but is compensated for by much improved protein and nucleic acid integrity.

In our hands, sections from AZF-fixed paraffin-wax-embedded BMT specimens are optimal for Giemsa, silver and Perl’s stains
(fig 1). The process of decalcification reduces the amount of stainable iron in BMT specimens. Previous studies have reported
that processing of BMT specimens with zinc–formalin fixative and formic acid decalcification results in an underestimation
of marrow iron. It is only in undecalcified material with plastic sections that ring sideroblasts are easily identified.22,23

Because of the nature of BMT samples received at the Hammersmith Hospital (predominantly neoplastic) and treatment or management
policies, we use immunohistochemical analysis on most cases (fig 2). Table 1 provides the details of the antibodies we use
on BMT specimens. In suspected cases of myeloproliferative disorders and myelodysplastic syndromes, we routinely carry out
CD34, CD117, ret40f, myeloperoxidase and CD42b/CD61. This helps us quantify the precursor cell population, identify foci of
abnormal localisation of immature precursor cells and identify subtle dysplastic features in the various lineages, including
identification of micromegakaryocytes.24–26 In addition, cases of systemic mastocytosis are immunostained for mast cell tryptase, CD25 and CD2.27,28 Cases of suspected acute leukaemia are investigated on a panel of antibodies that includes CD34, TdT, HLA-DR, CD99, myeloperoxidase,
CD117, CD68 (KP-1 and PG-M1), ret40f, CD61/CD42b, CD20, CD79a, CD3 and CD10.6,29–34 As well as helping in the proper classification of these cases, documentation of the leukaemia immunophenotype is of utmost
importance in evaluating follow-up BMT specimens for response to treatment, residual disease and early relapse.

On BMT specimens taken for staging histologically documented B cell non-Hodgkin’s lymphoma, we use a limited panel for immunohistochemistry—CD20,
CD3 and CD79a.35 In cases of patients with B cell non-Hodgkin’s lymphoma who have received anti-CD20 treatment, immunostaining with CD20 is
unreliable and may even be negative in residual B cell lymphoma cells. In such cases, CD79a is valuable in identifying residual
lymphoma. For staging BMT specimens in patients with anaplastic large-cell lymphomas, the panel includes CD20, CD3, CD30,
ALK, epithelial membrane antigen and Pax-5.36 The panel for staging BMT specimens of other node-based peripheral T cell lymphomas includes CD3, CD20, CD4, CD8, CD30, CD10
and CD21.6,37 In BMT specimens taken for staging Hodgkin’s lymphoma and in those where involvement is suspected on the H&E stain, the panel
includes CD20, CD3, CD15 and CD30. In difficult cases, an extended panel is required.

Myeloproliferative disorder; Mcm-2 in nucleus (red) and CD61 in cytoplasm of megakaryocytes (blue; ×400). The figure has
been contributed by Dr I Lampert and is part of his ongoing research.

Over the past 6 months, we have been carrying out ISH analysis of mRNA for κ and λ light chains and of EBV-encoded small RNA-1
for EBV. The results have been good and mRNA preservation has been more than satisfactory (fig 4). Although there have been
many studies investigating clonality in lymphoid or plasma cell proliferations with mRNA-ISH for immunoglobulin light chains,
only a few of the published studies have been on BMT specimens.47–49 Generally, immunostaining for light chains is adequate. In BMT samples, however, in cases which have abundant background
staining owing to adsorbed immunoglobulin in the background tissues, ISH results are often cleaner and much easier to interpret.
Few studies relating to ISH for EBV on BMTs exist. These can be of great value in identifying bone marrow involvement by EBV-associated
lymphoid malignancies such as T cell or natural killer cell lymphomas and Hodgkin’s lymphoma.50 Although cells expressing CD30 or CD3 or other tumour cell-marking antigens would not be restricted to the tumour cell population
in the marrow sample, EBV would almost be restricted to tumour cells. ISH for EBV-encoded small RNA-1 is more useful than
immunostaining for EBV-LMP-1 as it is more sensitive and is expressed across all types of EBV latency.51 The strategy would be very valuable in evaluating bone marrow samples from patients with HIV in whom 100% of cases of Hodgkin’s
lymphoma are EBV associated and the bone marrow is affected in a large proportion of patients. In our experience and that
of others, bone marrow may often be the initial site of disease presentation.52

More recently, we extracted DNA from BMT specimens processed by our protocol for the purposes of PCR amplification. We carried
out PCR analysis for immunoglobulin gene heavy-chain rearrangement on a few samples (fig 5). We need to study cases for a
wider array of PCR-based molecular diagnostics and analyse a large number of samples. Although many investigators find formalin-fixed,
EDTA-decalcified paraffin-wax-embedded BMT specimens to be adequate for molecular diagnostics, the preservation of DNA has
been previously reported to be superior with zinc-based fixatives compared with formalin.53,54

Laboratory integration and turnaround time

We aimed at making a protocol that would be quick, so as not to hamper the turnaround time and at the same time include procedures
that would generally be similar to other tissue processing protocols in the laboratory. The H&E-stained section with Giemsa,
reticulin and Perl’s and the initial panel of immunostains is ready in 72 h of receipt of BMT samples. The H&E-stained section
and the sections stained with histochemical stains are available in 48 h, if there is a need for urgent or provisional report
(table 2).

Turnaround time for bone marrow trephine biopsy specimens including first panel of immunohistochemistry

It should be emphasised that thin sectioning, preferably by an experienced histotechnologist, is a key variable in ensuring
excellent slides from which a maximal amount of diagnostic information can be extracted.

Affordability and toxicity

The costs of the reagents and their disposal are comparable with standard approaches and are considerably cheaper than resin-embedding
techniques. The AZF fixative is less toxic than many “special” fixatives and on a par with formalin. One of the major advantages
is that the processing is similar to that of a non-osseous biopsy specimen and can be incorporated into the routine laboratory
schedules.

CONCLUSIONS

Although we find that our method works very well at the Hammersmith Hospital, the ideal method for handling BMT specimens
should be worked out at each institution. Variables to be considered are the ability to provide haematologists with special
fixatives, the costs of fixatives (and the disposal thereof) and special processing such as resin embedding, the requirement
for rapid generation of diagnostic slides to speed turnaround time, the experience of the histotechnologists and the need
for molecular studies on the types of trephine biopsy specimens obtained at that institution. In addition, different fixation
and decalcification methods may have variable success in interacting with different types of tissue processors, antigen-retrieval
methods and immunohistochemistry methods. We believe that, with minimal adaptations to suit minor differences in general tissue
processing among different histopathology laboratories, the Hammersmith protocol described in this communication can be easily
standardised and maintained in any laboratory. This method provides excellent morphology and immunohistochemistry results.
In addition, the method is also optimal for preservation of nucleic acids, both DNA and RNA, for molecular diagnostics and
ISH-based studies.

Take-home messages

The Hammersmith Protocol provides a comprehensive approach to handle and investigate bone marrow trephine biopsy samples with
an intent to aid and affect management of haematological conditions, especially haematological malignancies.

The protocol provides excellent morphology, optimal antigen preservation for a complete array of immunohistochemistry and
nucleic acid preservation for PCR and mRNA in situ hybridisation-based studies.

Morphology is comparable to plastic sections as 1-μm-thin sections can be made.

The protocol can be used in a routine histopathology setup and does not require any specialised equipment.

The protocol provides a fast turnaround time with results of haematoxylin and eosin and other histochemical stains being available
in 48 h and those of other immunostains being available in 72 h of carrying out the biopsy.